Treatment for atherosclerosis and other cardiovascular and inflammatory diseases

ABSTRACT

Dithiocarboxylates, and in particular, dithiocarbamates, block the induced expression of the endothelial cell surface adhesion molecule VCAM-1, and are therefor useful in the treatment of cardiovascular disease, including atherosclerosis, post-angioplasty restenosis, coronary artery diseases, and angina, as well as noncardiovascular inflammatory diseases that are mediated by VCAM-1.

BACKGROUND OF THE INVENTION

This application is in the area of methods and compositions for thetreatment of atherosclerosis and other cardiovascular and inflammatorydiseases.

Adhesion of leukocytes to the endothelium represents a fundamental,early event in a wide variety of inflammatory conditions, includingatherosclerosis, auto immune disorders and bacterial and viralinfections. This process is mediated in part by the induced expressionof endothelial cell surface adhesion molecules, such as ICAM-1(intracellular adhesion molecule-1), VCAM-1 (vascular adhesionmolecule-1) and ELAM-1 (endothelial leukocyte adhesion molecule-1).These adhesion molecules bind to immune cells, which initiate andpropagate the inflammatory response. One of the adhesion molecules,VCAM-1, plays an especially important role in binding monocytes.Multiple signals induce the expression of the cell surface adhesionmolecules.

Atherosclerosis is a chronic inflammatory disease of the arterial intimacharacterized by the focal accumulation of leukocytes, smooth musclecells, lipids and extracellular matrix. A central feature, and one ofthe earliest detectable events, in the pathogenesis of atheroscleroticplaque is the adherence of mononuclear leukocytes to discrete segmentsof the arterial endothelium through the VCAM-2 proteins on the surfaceof the vascular endothelial cells. After attachment to the endothelialcells, the mononuclear leukocytes are transformed into lipid-ladenmacrophages, or "foam cells" Atherosclerosis begins as a highly focallesion within the vascular wall usually in areas where the normal,laminar flow of blood is disturbed such as at vessel flow dividers.These "low shear stress" areas are characterized by an abnormal Localaccumulation of oxidatively-modified low density lipoprotein (ox-LDL).

It has been suggested that early events in the pathogenesis ofatherosclerosis are mediated by low density lipoprotein that has beenconverted by reactive oxygen species into ox-LDL. Steinberg, et al.,"Beyond cholesterol: Modifications of low-density lipoprotein thatincrease its atherogenicity." N. Engl. J. Med. 320:915-924 (1989);Parthasarathy, et al., "Probucol inhibits oxidative modification of lowdensity lipoprotein." J. Clin. Invest 77(2):641-4 (1986). It is notclear by what mechanism LDL is oxidized either intracellularly orextracellularly.

Current therapies for cardiovascular disease, and in particular,atherosclerosis do not treat the cause of the disease, but instead treatthe symptoms of the disease or lower risk factors associated with thedisease. Pharmaceutical agents prescribed for these conditions includelipid lowering agents such as probucol and nicotinic acid; aspirin(which prevents platelets from sticking); antithrombotic agents such ascoumadin; calcium channel blockers such as varapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalopril, and β-blockers such as propanalol, terbutalol,and labetalol. Since the therapeutic agents are not selective, theyaffect many different organs, and have significant side effects. Thereare currently no drugs that are directed at inhibiting the binding ofmonocytes to cell surface adhesion molecules such as VCAM-1.

Given that cardiovascular disease is currently the leading cause ofdeath in the United States, and ninety percent of cardiovascular diseaseis presently diagnosed as atherosclerosis, there is a strong need toidentify new methods and pharmaceutical agents for their treatment.

Dithiocarbamates are transition metal chelators clinically used forheavy metal intoxication. Baselt, R. C., F. W. J. Sunderman, et al.(1977), "Comparisons of antidotal efficacy of sodiumdiethyldithiocarbamate, D-penicillamine and triethylenetetramine uponacute toxicity of nickel carbonyl in rats." Res Commun Chem PatholPharmacol 18(4):677-88; Menne, T. and K. Kaaber (1978), "Treatment ofpompholyx due to nickel allergy with chelating agents." ContactDermatitis 4(5): 289-90; Sunderman, F. W. (1978), "Clinical response totherapeutic agents in poisoning from mercury vapor" Ann Clin Lab Sci8(4): 259-69; Sunderman, F. W. 1979), "Efficacy of sodiumdiethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning."Ann Clin Lab Sci 9(1): 1-10; Gale, G. R., A. B. Smith, et al. (1981) ,"Diethyldithiocarbamate in treatment of acute cadmium poisoning." AnnClin Lab Sci 11(6): 476-83; Jones, M. M. and M. G. Cherian (1990), "Thesearch for chelate antagonists for chronic cadmium intoxication."Toxicology 62(1): 1-25; Jones, S. G., M. A. Basinger, et al. (1982) , "Acomparison of diethyldithiocarbamate and EDTA as antidotes for acutecadmium intoxication." Res Commun Chem Pathol Pharmacol 38(2): 271-8;Pages, A., J. S. Casas, et al. (1985), "Dithiocarbamates in heavy metalpoisoning: complexes of N,N-di(1-hydroxyethyl)dithiocarbamate withZn(II), Cd(II), Hg(II), CH3Hg(II), and C6HSHg(II): J. Inorg Biochem25(1): 35-42; Tandon, S. K., N. S. Hashmi, et al. (1990), "Thelead-chelating effects of substituted dithiocarbamates." Biomed EnvironSci 3(3): 299-305.

Dithiocarbamates have also been used adjunctively in cis-platinumchemotherapy to prevent renal toxicity. Hacker, M. P., W. B. Ershler, etal. (1982) . "Effect of disulfiram (tetraethylthiuram disulfide) anddiethyldithiocarbamate on the bladder toxicity and antitumor activity ofcyclophosphamide in mice." Cancer Res 42(11): 4490-4. Bodenner, 1986#733 Chelation of transition metals would have the effect of blockinghydroxyl radical production intracellular via the Haber-Weiss-Fentonreaction). Saran, M. and Bors, W. (1990). "Radical reactions in vivo--anoverview." Radiat. Environ. Biophys. 29(4):249-62.

A dithiocarbamate currently used in the treatment of alcohol abuse isdisulfiram, a dimer of diethyldithiocarbamate. Disulfuram inhibitshepatic aldehyde dehydrogenase. Inoue, K., and Fukunaga, et al., (1982)."Effect of disulfiram and its reduced metabolite, diethyldithiocarbamateon aldehyde dehydrogenase of human erythrocytes." Life Sci 30(5):419-24.

It has been reported that dithocarbamates inhibit HIV virus replication,and also enhance the maturation of specific T cell subpopulations. Thishas led to clinical trials of diethyldithiocarbamate in AIDs patientpopulations. Reisinger, E., et al., (1990). "Inhibition of HIVprogression by dithiocarb." Lancet 335:679.

It is therefore an object of the present invention to provide a methodfor the treatment of atherosclerosis and other cardiovascular andinflammatory diseases.

It is another object of the present invention to provide pharmaceuticalcompositions for the treatment of atherosclerosis and othercardiovascular and inflammatory diseases.

It is still another object of the present invention to provide methodsand compositions to block the ability of cells to express gene productsknown to be responsible for the adherence of leukocytes to those cells,and activation of the cells.

SUMMARY OF THE INVENTION

It has been discovered that dithiocarboxylates, and in particular,dithiocarbamates, block the induced expression of the endothelial cellsurface adhesion molecule VCAM-1, and thus are useful in the treatmentof atherosclerosis, post-angioplasty restenosis, coronary arterydiseases, angina, and other cardiovascular diseases, as well asnoncardiovascular inflammatory diseases that are mediated by VCAM-1.

Importantly, certain dithiocarbamates, such as sodium pyrrolidineN-dithiocarbamate (PDTC), do not significantly block the inducedexpression of other endothelial cell surface adhesion molecules, such asICAM-1 or ELAM-1, and therefore, do not adversely affect inflammatoryresponses that VCAM-1 does not mediate. It has also been discovered thatPDTC exhibits preferential toxicity to proliferating or abnormallydividing vascular smooth muscle cells. Another dithiocarbamate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, also inhibits the expressionof VCAM-1, without significant effect on ICAM-1, but does not exhibitpreferential toxicity to abnormally dividing vascular smooth musclecells. The ability of the other active dithiocarbamates to selectivelyinhibit the expression of VCAM-1 (without inhibiting the expression ofELAM-1 or ICAM-1), and to exhibit preferential toxicity to abnormallydividing smooth muscle cells, is evaluated according to methodsdisclosed herein.

Dithiocarbamates that are useful in the treatment of atherosclerosis andother cardiovascular and inflammatory diseases include, but are notlimited to, those of the formulas:

    R.sup.1 SC(S)NR.sup.2 R.sup.3 or R.sup.2 R.sup.3 N(S)CS--SC(S)NR.sup.2 R.sup.3

wherein R¹ is H or a pharmaceutically acceptable cation, including butnot limited to sodium, potassium, or NR⁴ R⁵ R⁶ R⁷, wherein R⁴, R⁵, R⁶,and R⁷ are independently hydrogen, C₁₋₆ linear, branched, or cyclicalkyl, hydroxy(C₁₋₆)alkyl (wherein one or more hydroxyl groups arelocated on any of the carbon atoms), or aryl, and

R² and R³ are independently C₁₋₁₀ linear, branched or cyclic alkyl;--(CHOH)_(n) (CH₂)_(n) OH, wherein n is 0, 1, 2, 3, 4, 5, or 6;--(CH₂)_(n) CO₂ R¹, --(CH₂)_(n) CO₂ R⁴ ; hydroxy(C₁₋₆)alkyl--, or R² andR³ together constitute a bridge such as --(CH₂)_(m) --, wherein m is3-6, and wherein R⁴ is alkyl, aryl, alkaryl, or aralkyl, includingacetyl, propionyl, and butyryl.

The active dithiocarboxylates, and in particular dithiocarbamates,disclosed herein can be used in the treatment of acute and chronicinflammatory diseases mediated by VCAM-1, including but not limited torheumatoid and osteoarthritis, asthma, dermatitis, and may be of benefitin the treatment of multiple sclerosis.

The compounds are useful in both the primary and adjunctive medicaltreatment of cardiovascular disease. The compounds can be used asadjunctive therapy in combination with agents administered to reduce therisk of disease by lowering LDL and serum cholesterol. The methodrepresents a significant advance in treating cardiovascular disease, inthat it goes beyond the current therapies designed simply to inhibit theprogression of the disease, and when used appropriately, provides thepossibility to medically "cure" atherosclerosis by preventing newlesions from developing and causing established lesions to regress. Thecompounds can be administered to treat small vessel disease that is nottreatable by surgery or angioplasty, or other vessel disease in whichsurgery is not an option. The compounds can also be used to stabilizepatients prior to revascularization therapy.

The active compound or a mixture of the compounds are administered inany appropriate manner, including but not limited to orally andintravenously. General range of dosage will be from 0.5 to 500 mg/kgbody weight with a dose schedule ranging from once every other day totwice a day. The length of dosing will range from a single dose givenonly once to twice daily to dosages given over the course of two to sixmonths.

The compounds can also be administered directly to the vascular wallusing perfusion balloon catheters following or in lieu of coronary orother arterial angioplasty. As an example, 2-5 mL of a physiologicallyacceptable solution that contains approximately 1 to 500 μM of thecompound or mixture of compounds is administered at 1-5 atmospherespressure. Thereafter, over the course of the next six months during theperiod of maximum risk of restenosis, the active compounds areadministered through other appropriate routes and dose schedules.

Relatively short term treatments with the active compounds are used tocause the "shrinkage" of coronary artery disease lesions that cannot betreated either by angioplasty or surgery. A nonlimiting example of shortterm treatment is two to six months of a dosage ranging from 0.5 to 500mg/kg body weight given at periods ranging from once every other day tothree times daily.

Longer term treatments can be employed to prevent the development ofadvanced lesions in high-risk patients. A long term treatment can extendfor years with dosages ranging from 0.5 to 500 mg/kg body weightadministered at intervals ranging from once every other day to threetimes daily.

The active compounds can also be administered in the period immediatelyprior to and following coronary angioplasty as a means to reduce oreliminate the abnormal proliferative and inflammatory response thatcurrently leads to clinically significant restenosis.

The active compounds can be administered in conjunction with othermedications used in the treatment of cardiovascular disease, includinglipid lowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as varapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalopril, and β-blockers such as propanalol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteroidal antiinflammatories such as ibuprofen, indomethacin,fenoprofen, mefenamic acid, flufenamic acid, sulindac, or withcorticosteriods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of an autoradiogram of mRNA, obtained asdescribed below, hybridized to either ³² p-labeled human VCAM-1 specificcDNA (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1specific cDNA (Panel C). Following pre-treatment for 30 minutes with 50μM of sodium pyrrolidine dithiocarbamate (PDTC), HUVE (human umbilicalvein) cells were exposed to IL-lb (10 U/ml) in the continuous presenceof 50 μM PDTC. Parallel controls were performed identically except inthe absence of PDTC. At the indicated times, total RNA was isolated and20 μg of material size-fractionated by denaturing 1.0%agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose,hybridized as described above, and visualized by autoradiography. Lane1--0 hour; Lanes 2,4,6,8--OL-1 alone for 2, 4, 8 and 24 hours,respectively; Lanes 3,5,7,9--IL-1 with PDTC for 2,4,8 and 24 hours,respectively.

FIG. 2 is an illustration of an autoradiogram of mRNA, obtained asdescribed below, hybridized to either ³² p-labeled human VCAM-1 specific(Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1specific cDNA (Panel C). HUVE cells were pretreated with the indicatedconcentrations of PDTC, and then exposed to IL-lb in the presence ofPDTC for four hours and assayed for VCAM-1 mRNA accumulation by Northernfilter hybridization analysis. Lane 1--control, lane 2--IL-1 (10 u/ml),lane 3--IL-lb+PDTC (0.05 μM), lane 4--IL-1 LB+PDTC (0.5 μM), lane5--IL-lb+PDTC (5.0 μM), lane 6--IL=lb+PDTC (50.0 μM), lane 7--IL-lb+PDTC(100 μM).

FIG. 3 is an illustration of an autoradiogram of mRNA, obtained asdescribed below, hybridized to either ³² p-labeled human VCAM-1 specificcDNA (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1specific cDNA (Panel C). HUVE cells were pretreated as described in FIG.1 with 50 μM PDTC, exposed for four hours to the agents indicated below,and assayed for VCAM-1 (Panel A) and ICAM-1 (Panel B) mRNA accumulation.Lane 1--TNFa (100 U/ml), lane 2--TNFa+PDTC, lane 3--lipopolysaccharide(LPS) (100 ng/ml), lane 4--LPS+PDTC, lane 5--poly(I:C) (100 mg/ml), lane6--poly(I:C)+PDTC.

FIG. 4 is a graph of relative cell surface expression of VCAM-1 andICAM-1 in the presence (dark bars) or absence (white bars) of PDTC andin the presence of multiple types of inducing stimuli. Confluent HUVECswere pretreated or not pretreated (CTL only) for 30 minutes with 50 μMPDTC, and then exposed for the indicated times to the indicated agentsin the presence or absence (CTL only) of PDTC. Cell surface expressionwas determined by primary binding with VCAM-1 specific (4B9) and ICAM-1specific (84H10) mouse monoclonal antibodies followed by secondarybinding with a horse-radish peroxidase tagged goat anti-mouse (IgG).Quantitation was performed by determination of calorimetric conversionat 450 nm of TMB. FIG. 4 indicates that multiple regulatory signalsinduce VCAM-1 but not ICAM-1 through a common, dithiocarbamate-sensitivepathway in human vascular endothelial cells.

FIG. 5 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in human umbilical vein endothelial cells, activated by TNFa,versus concentration of various antioxidants. (PDTC is sodiumN-pyrrolidine dithiocarbamate; DETC is sodiumN,N-diethyl-N-carbodithiolate, also referred to as sodiumdiethyldithiocarbamate; NAC is N-acetyl cysteine; and DF isdesferroximine).

FIG. 6 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in human umbilical vein endothelial cells, activated by TNFa, inthe presence of the specified amount of antioxidant. (PDTC is sodiumN-pyrrolidine dithiocarbamate; DIDTC is sodiumN,N-diethyl-N-carbodithioate; SarDTC is sodiumN-methyl-N-carboxymethyl-N-carbodithioate; IDADTC is trisodiumN,N-di(carboxymethyl)-N-carbodithioate; MGDTC is sodiumN-methyl-D-glucamine-N-carbodithioate; MeOBGDTC is sodiumN-(4-methoxybenzyl)-D-glucamine-N-carbodithioate; DEDTC is sodiumN,N-diethyl-N-carbodithioate; Di-PDTC is sodiumN,N-diisopropyl-N-carbodithioate; NAC is N-acetyl cysteine.)

FIG. 7 is a graph of the percentage of Molt-4 cells binding to HUVEcells either unstimulated or stimulated with TNFa (100 U/ml) for sixhours in the presence or absence of PDTC.

FIG. 8 is an illustration of the chemical structures of the followingactive dithiocarbamates: sodium pyrrolidine-N-carbodithioate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, trisodiumN,N-di(carboxymethyl)-N-carbodithioate, sodiumN-methyl-D-glucamine-N-carbodithioate, sodiumN,N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate), and sodiumN,N-diisopropyl-N-carbodithioate.

DETAILED DESCRIPTION OF THE INVENTION

The term alkyl, as used herein, unless otherwise specified, refers to asaturated straight, branched, or cyclic hydrocarbon of C₁ to C₁₀, andspecifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

The term alkenyl, as referred to herein, and unless otherwise specified,refers to a straight, branched, or cyclic hydrocarbon of C₂ to C₁₀ withat least one double bond.

The term alkynyl, as referred to herein, and unless otherwise specified,refers to a C₂ to C₁₀ straight or branched hydrocarbon with at least onetriple bond.

The term aralkyl refers to an aryl group with at least one alkylsubstituent.

The term alkaryl refers to an alkyl group that has at least one arylsubstituent.

The term halo (alkyl, alkenyl, or alkynyl) refers to an alkyl, alkenyl,or alkynyl group in which at least one of the hydrogens in the group hasbeen replaced with a halogen atom.

The term aryl, as used herein, and unless otherwise specified, refers tophenyl or substituted phenyl, wherein the phenyl ring has one or more ofthe following substituents: hydroxy, CO₂ H, or its pharmaceuticallyacceptable salt, CO₂ (alkyl), alkoxy, alkyl or glucamine.

The term alkoxy, as used herein, and unless otherwise specified, refersto a moiety of the structure --O-alkyl.

The term halo, as used herein, includes fluoro, chloro, bromo, and iodo.

The term hydroxyalkyl, as used herein, refers to a C₁ to C₆ alkyl groupin which at least one of the hydrogens attached to any of the carbonatoms is replaced with a hydroxy group.

The term thiol antioxidant refers to a sulfur containing compound thatretards oxidation.

The term pharmaceutically acceptable derivative refers to a derivativeof the active compound that upon administration to the recipient, iscapable of providing directly or indirectly, the parent compound, orthat exhibits activity itself.

I. Active Compounds

It has been discovered that dithiocarboxylates are useful in thetreatment of atherosclerosis and other cardiovascular and inflammatorydiseases. Dithiocarboxylates, including dithiocarbamates, can be used toblock the ability of cells, including endothelial cells, to expressVCAM-1, which is a gene product known to be responsible for theadherence of leukocytes to cells. The fact that dithiocarboxylates,including dithiocarbamates, inhibit VCAM-1 gene expression stronglysupports the importance of oxidation as an initiating signal in alteredvascular-inflammatory cell interactions. The specific molecularmechanisms by which the dithiocarboxylates function in inhibiting VCAM-1gene expression is not known.

At least one of the compounds, sodium pyrrolidine dithiocarbamate(PDTC), inhibits VCAM-1 gene expression at a concentration of less than1.0 micromolar. This compound also exhibits preferential toxicity toproliferating or abnormally dividing vascular smooth muscle cells. Ithas been discovered that sodium pyrrolidine dithiocarbonate does notsignificantly block ELAM-1 or ICAM-1 expression, and therefore treatmentwith this compound does not adversely affect aspects of the inflammatoryresponse mediated by ELAM-1 or ICAM-1. Thus, a generalizedimmunosuppression is avoided. This may avoid systemic complications fromgeneralized inhibition of adhesion molecules in the many other celltypes known to express them.

Dithiocarboxylates are compounds of the structure A-SC(S)-B, which aremembers of the general class of compounds known as thiol antioxidants,and are alternatively referred to as carbodithiols or carbodithiolates.It appears that the SC(S) moiety is essential for therapeutic activity,and that A and B can be any group that does not adversely affect theefficacy or toxicity of the compound. A and B can be selected by one ofordinary skill in the art to impart desired characteristics to thecompound, including size, charge, toxicity, and degree of stability(including stability in an acidic environment such as the stomach, orbasic environment such as the intestinal tract). The selection of A andB will also have an important effect on the tissue-distribution andpharmacokinetics of the compound. In general, for treatment ofcardiovascular disease, it is desirable that the compound accumulate, orlocalize, in the arterial intimal layer containing the vascularendothelial cells. The compounds are preferably eliminated by renalexcretion.

As nonlimiting examples, A and B can be independently alkyl, alkenyl,alkynyl, alkaryl, aralkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl,alkaryl, hydrogen, C₁₋₆ alkoxy-C₁₋₁₀ alkyl, C₁₋₆ alkythio-C₁₋₁₀ alkyl,C₁₋₁₀ substituted alkyl (wherein the substituent is independentlyhydroxy, carbonyl, or carboxylic acid, located on any of C₁₋₁₀), NR² R³,--(CHOH)_(n) CH₂ OH, wherein n is 0, 1, 2, 3, 4, 5, or 6, --(CH₂)_(n)CO₂ R¹, including acetyl, propionyl, and butyryl, hydroxy(C₁₋₆)alkyl--wherein one or more hydroxyl groups are located on any ofthe carbon atoms), and A can be a pharmaceutically acceptable cation,including but not limited to sodium, potassium, or NR⁴ R⁵ R⁶ R⁷. In analternative embodiment, a dimer such as B--C(S)S--SC(S)--B can beadministered.

Dithiocarboxylates should be chosen for use in treating atherosclerosisand other cardiovascular and inflammatory diseases that have the properlipophilicity to locate at the affected site. The compound should notcompartmentalize in low turnover regions such as fat deposits. In apreferred embodiment for treatment of cardiovascular disease, thepharmacokinetics of the compound should not be dramatically affected bycongestive heart failure or renal insufficiency.

The dithiocarboxylate must be physiologically acceptable- In general,compounds with a therapeutic index of at least 2, and preferably atleast 5 or 10, are acceptable. The therapeutic index is defined as theEC₅₀ /IC₅₀, wherein EC₅₀ is the concentration of compound that inhibitsthe expression of VCAM-1 by 50% and IC₅₀ is the concentration ofcompound that is toxic to 50% of the target cells. Cellular toxicity canbe measured by direct cell counts, trypan blue exclusion, or variousmetabolic activity studies such as 3H-thymidine incorporation, as knownto those skilled in the art. The therapeutic index of PDTC in tissueculture is over 100 as measured by cell toxicity divided by ability toinhibit VCAM-1 expression activated by TNFa, in HUVE (human umbilicalvein) cells. Initial studies on the rapidly dividing cell type HT-18human glioma demonstrate no toxicity at concentrations 100-fold greaterthan the therapeutic concentration. Disulfiram, an orally administeredform of diethyldithiocarbamate, used in the treatment of alcohol abuse,generally elicits no major clinical toxicities when administeredappropriately.

There are a few dithiocarbamates that are known to be genotoxic. Thesecompounds do not- fall within the scope of the present invention, whichis limited to the administration of physiologically acceptablematerials. An example of a genotoxic dithiocarbamate is the fungicidezinc dimethyldithiocarbamate. Further, the anticholinesterase propertiesof certain dithiocarbamates can lead to neurotoxic effects. Miller, D.(1982). Neurotoxicity of the pesticidal carbamates. Neurobehav. Toxicol.Teratol. 4(6): 779-87.

The term dithiocarboxylate as used herein specifically includes, but isnot limited to, dithiocarbamates of the formulas:

    R.sup.1 SC(S)NR.sup.2 R.sup.3 and R.sup.2 R.sup.3 N(S)CS--SC(S)NR.sup.2 R.sup.3

wherein R¹ is H or a pharmaceutically acceptable cation, including butnot limited to sodium, potassium, or NR⁴ R⁵ R⁶ R⁷, wherein R⁴, R⁵, R⁶,and R⁷ are independently hydrogen, C₁₋₆ linear, branched, or cyclicalkyl, hydroxy(C₁₋₆)alkyl (wherein one or more hydroxyl groups arelocated on any of the carbon atoms), or aryl, and

R² and R³ are independently C₁₋₁₀ linear, branched or cyclic alkyl;--(CHOH)_(n) (CH₂)_(n) OH, wherein n is 0, 1, 2, 3, 4, 5, or 6;--(CH₂)_(n) CO₂ R¹, --(CH₂)_(n) CO₂ R⁴ ; hydroxy(C₁₋₆)alkyl--, or R² andR³ together constitute a bridge such as --(CH₂)_(m) --, wherein m is3-6, and wherein R⁴ is alkyl, aryl, alkaryl, or aralkyl, includingacetyl, propionyl, and butyryl.

Specific examples of useful dithiocarbamates, illustrated in FIG. 8,include sodium pyrrolidine-N-carbodithioate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, trisodiumN,N-di(carboxymethyl)-N-carbodithioate, sodiumN-methyl-D-glucamine-N-carbodithioate, sodiumN,N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate), and sodiumN,N-diisopropyl-N-carbodithioate.

The active dithiocarboxylates and in particular, dithiocarbamates areeither commercially available or can be prepared using known methods.

At the molecular level, PDTC has been shown to inhibit the activation ofthe transcriptional regulatory factor Nf-kB in response to certaincytokine and non-cytokine stimuli (Schreck, Rieber et al. 1991; Schreck,Meier et al. 1992). However, by gel mobility shift assay of HUVECnuclear extracts with various kb-like enhancer motifs, it has beendiscovered that endothelial cells activate VCAM-1 gene expressionthrough an apparently novel transcriptional regulatory factor that isnot Nf-kB. This suggests that PDTC may regulate endothelial cell geneexpression through its effect on a new transcriptional regulatoryprotein. It has also been demonstrated that VCAM-1 expression is inducedby multiple factors in cultured Kaposi's sarcoma cells, which may beimportant in its pathogenesis. PDTC blocks VCAM-1 expression in Kaposi'ssarcoma cells that have been activated by TNF, IL-1, and poly(I:C).

It has also been discovered that smooth muscles cells produce a solubleform of VCAM-1 which may be secreted by the cells, and which may act asa natural antihistamine.

II. Biological Activity

The ability of dithiocarboxylates to inhibit the expression of VCAM-1can be measured in a variety of ways, including by the methods set cutin detail below in Examples 1 to 7. For convenience, Examples 1-3 and6-7 describe the evaluation of the biological activity of sodiumPyrrolidine-N-carbodithioate (also referred to as PDTC). These examplesare not intended to limit the scope of the invention, which specificallyincludes the use of any of the above-described compounds to treatatherosclerosis, and other types of inflammation and cardiovasculardisease mediated by VCAM-1. Any of the compounds described above can beeasily substituted for PDTC and evaluated in similar fashion.

Examples 4 and 5 provide comparative data on the ability of a number ofdithiocarbamates to inhibit the gene expression of VCAM-1. The examplesbelow establish that the claimed dithiocarbamates specifically block theability of VCAM-1 to be expressed by vascular endothelial cells inresponse to many signals known to be active in atherosclerosis and theinflammatory response.

Experimental Procedures

Cell Cultures

HUVE cells were isolated from human umbilical veins that werecannulated, perfused with Hanks solution to remove blood, and thenincubated with 1% collagenase for 15 minutes at 37° C. After removal ofcollagenase, cells were cultured in M199 medium supplemented with 20%fetal bovine serum (HyClone), 16 μg/ml heparin (ESI Pharmaceuticals,Cherry Hill, N.J.), 50 μg/ml endothelial cell growth supplement(Collaborative Research Incorporated, Bedford Ma.), 25 mM Hepes Buffer,2 mM L-glutamin, 100 μg/ml penicillin and 100 μg/ml streptomycin andgrown at 37° C. on tissue culture plates coated 0.1% gelatin. Cells werepassaged at confluency by splitting 1:4. Cells were used within thefirst 8 passages.

Incubation with Cytokines and Other Reagents

Confluent HUVE cells were washed with phosphate buffered saline and thenreceived fresh media. The indicated concentrations of PDTC were added aspretreatment 30 minutes before adding cytokines. Cytokines and otherinducers were directly added to medium for the times and at theconcentrations indicated in each experiment. Human recombinant IL-lb wasthe generous gift of Upjohn Company (Kalamazoo, Mich.). TNFa wasobtained from Bohringer Engelheim. Bacterial lipopolysaccharide (LPS),polyinosinic acid: polycitidilic acid (Poly I:C), and pyrrolidinedithiocarbarnate (PDTC) were obtained from Sigma Chemical (St. Louis,Mo.). All other reagents were of reagent grade.

RNA Isolation

Total cellular RNA was isolated by a single extraction using an acidguanidium thiocyanate-phenol-chloroform mixture. Cells were rinsed withphosphate buffered saline and then lysed with 2 ml of guanidiumisothiocyanate. The solution was acidified with 0.2 ml of sodium acetate(pH 4.0) and then extracted with 2 ml phenol and 0.4 mlchloroform:isoamyl alcohol (24:1). The RNA underwent two ethanolprecipitations prior to being used for Northern blot analysis.

Northern Blot Analysis

Total cellular RNA (20 μg) was size fractionated using 1% agaroseformaldehyde gels in the presence of 1 μg/ml ethidium bromide. The RNAwas transferred to a nitrocellulose filter and covalently linked byultraviolet irradiation using a Stratlinker UV crosslinker (Stratagene,La Jolla, Ca.). Hybridizations were performed at 42° C. for 18 hours in5× SSC (1X=150 mM NaCl, 15 mM Na citrate), 1% sodium dodecyl sulfate, 5×Denhardt solution, 50% formamide, 10% dextran sulfate and 100 μg/ml ofsheared denatured salmon sperm DNA. Approximately 1-2×10⁶ cpm/ml oflabeled probe (specific activity>10⁸ cpm/μg DNA) were used perhybridization. Following hybridization, filters were washed with a finalstringency of 0.2×SSC at 55° C. The nitrocellulose was stripped usingboiled water prior to rehybridization with other probes. Autoradiographywas performed with an intensifying screen at -70° C.

³² Probes

³² P labeled DNA probes were made using the random primeroligonucleotide method. The ICAM-1 probe was an Eco R1 fragment of humancDNA. The ELAM-1 probe was a 1.85 kb Hind III fragment of human cDNA.The VCAM-1 probe was a Hind III-Xho I fragment of the human cDNAconsisting of nucleotide 132 to 1814.

Enzyme Linked Immunosorbent Assay (ELISA)

HUVE cells were plated on 96-well tissue culture plates 48 to 72 hoursbefore the assay. Primary antibodies in M199 with 5% FBS were added toeach well and incubated one hour at 37° C. The cells were then washedand incubated for one hour with peroxidase conjugated goat anti-mouseIgG (Bio Rad) diluted 1/500 in M199 with 5% FBS. The wells were t-henwashed and binding of antibody was detected by the addition of 100 μl of10 mg/ml 3,3,5,5'-tetramethyl-benzidine (Sigma) with 0.003% H₂ O₂. Thereaction was stopped by the addition of 25 μl of 8N sulfuric acid.Plates were read on an ELISA reader (Bio Rad) at OD 450 nm afterblanking on rows stained only with second step antibody. Data representthe means of triplicate.

Antibodies

Monoclonal antibody (MAb) 4B9 recognizing vascular cell adhesionmolecule-1 (VCAM-1) was the generous gift of Dr. John Harlan (Universityof Washington). MAb E9A1F1 recognizing endothelial cell adhesionmolecule (ELAM-1) was the generous gift of Dr. Swerlick (EmoryUniversity). Hybridomas producing mAb 84H10 recognizing intercellularadhesion molecule 1 (ICAM-1) are routinely grown in our laboratory andantibody was used as tissue culture supernatant.

EXAMPLE 1

PDTC Blocks IL-lb Mediated Induction of HUVEC VCAM-1, but not ICAM-1 orELAM-1, mRNA Accumulation

To determine whether the oxidative state of the endothelial cell canalter the basal or induced expression of cell adhesion molecule genes,cultured human vascular endothelial cells were exposed to the inducingcytokine, IL-lb (10 U/ml) in the presence or absence of the thiolatedmetal chelating antioxidant, pyrrolidine dithiocarbamate (PDTC, 50 μM)for up to 24 hours. As shown in FIG. 1, IL-lb alone (lanes 2, 4, 6, 8)induces the expected rapid and transient induction of VCAM-1 (Panel A),E-selectin (ELAM-1, Panel B) and ICAM-1 (Panel C) mRNA accumulation, allof which peak at four hours. However, in the presence of PDTC, IL-lbinduction of VCAM-1 mRNA accumulation is dramatically inhibited by over90% (panel A, lanes 3, 5, 7, 9). In contrast, although IL-lb mediatedinduction of ELAM-1 is slightly inhibited at 2 and 24 hours (comparelane 2 and 3, 8 and 9, panel B), PDTC does not inhibit the induction at4 and 8 hours (lane 5 and 7, panel B). IL-lb mediated induction ofICAM-1 mRNA accumulation is not affected (panel B, lanes 3, 5, 7, 9).Indeed, a mild augmentation of IL-lb induction of ICAM-1 mRNAaccumulation (˜30%) is observed (compare lanes 4 and 5, panel B).Equivalent amounts of nitrocellulose transferred RNA per lane wasconfirmed by ethidium bromide staining and visualization.

A dose-response analysis was performed to determine whether PDTCinhibits the induction of VCAM-1 gene expression by IL-lb in a dosedependent manner. As shown in FIG. 2, PDTC inhibits IL-lb mediatedinduction of VCAM-1 gene expression with a steep dose-response curve(FIG. 2, panel A) with a calculated EC₅₀ of approximately 10 μM, whilePDTC does not inhibit IL-lb mediated induction of ELAM-1 expression withthese concentrations (FIG. 2, panel B). The IL-lb mediated induction ofICAM-1 mRNA accumulation is enhanced by PDTC with the concentrationhigher than 0.5 μM (FIG. 2, compare lane 2 and lane 4-7, panel C).

These data demonstrate that IL-lb utilizes a dithiocarboxylate, and inparticular, a dithiocarbamate sensitive step as part of its signalingmechanism in the induction of VCAM-1 gene expression. The data alsoappear to indicate that this dithiocarbamate sensitive step does notplay a significant role in the IL-lb mediated induction of ELAM-1 orICAM-1 gene expression.

EXAMPLE 2

PDTC Blocks Induction of HUVEC VCAM-1 mRNA Accumulation by MultipleStimuli

To determine whether other well-described activators of VCAM-1 geneexpression also utilize a PDTC sensitive step, three distinct classes ofactivators were tested: another classic receptor mediated inducing agent(tumor necrosis factor, TNFa), a non-receptor mediated inducer(lipopolysaccharide, LPS) and a recently described novel inducer (doublestranded RNA, poly(I:C)). In all three cases, PDTC dramaticallyinhibited the induction of VCAM-1 mRNA accumulation in HUVECs after fourhours (FIG. 3, Panel A). Although the TNFa mediated ELAM-1 geneexpression is suppressed to some extent (FIG. 3 lane 1 and 2, panel B),LPS and poly(I:C) mediated ELAM-1 mRNA accumulation was unaffected (FIG.3 lane 3-6, panel B). The induction of ICAM-1 mRNA accumulation wasunaffected (FIG. 3, Panel C). This data indicates that structurallydistinct inducing agents, acting through distinct pathways, share acommon regulatory step specific for the induction of VCAM-1 geneexpression.

EXAMPLE 3

PDTC Blocks HUVE Cell Surface Expression of VCAM-1 Induced by MultipleStimuli

To determine whether, like its mRNA, the induction of endothelial cellsurface protein expression of VCAM-1 could also be inhibited by PDTC,monoclonal antibodies were used in an ELISA assay to quantitate theinduction of cell surface VCAM-1 and ICAM-1 in cultured HUVE cells. Asshown in FIG. 4, multiple classes of activating agents, in the absenceof PDTC (--PDTC), induce the rapid and transient accumulation of VCAM-1(top left panel) at the cell surface peaking at six hours. In thepresence of PDTC (+PDTC, top right panel), the induction of cell surfaceexpression of VCAM-1 by all agents tested is dramatically inhibited(80-90%). In contrast, the induced expression of cell surface ICAM-1 isunaffected under identical conditions (bottom left and right panels).

These data demonstrate that, like its mRNA accumulation, cell surfaceVCAM-1 expression are selectively inhibited by dithiocarbamates and thatmultiple classes of activating agents utilize a similar, dithiocarbamatesensitive mechanism to induce VCAM-1 gene expression.

EXAMPLE 4

Comparative Effectiveness of Antioxidants in Blocking TNFa Induction ofVCAM-1

To determine whether structurally similar or dissimilar antioxidantscould also inhibit VCAM-1 gene expression, and with what potency, HUVEcells were exposed to TNFa for six hours in the presence or absence ofdifferent concentrations of four different antioxidants. As shown inFIG. 5, both diethyldithiocarbamate (DETC) and N-acetyl cysteine (NAC)inhibited VCAM-1 expression at concentrations of 5 μM and 30 μM,respectively. In contrast, PDTC (PDTC) was effective between 5 and 50μM. The iron metal chelator, desferroximine, had no effect at theconcentrations tested.

EXAMPLE 5

PDTC Inhibits TNF Induction of VCAM-1/VLA-4 Mediated Adhesion

The ability of a variety of antioxidants to inhibit TNFa induction ofVCAM-1 in HUVE cells was evaluated by the method set out in Example 4.FIG. 6 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in TNFa activated HUVE cells versus concentrations of PTDC(sodium N-pyrrolidine dithiocarbamate), DIDTC (sodiumN,N-diethyl-N-carbodithioate), SarDTC (sodiumN-methyl-N-carboxymethyl-N-carbodithioate), IDADTC (trisodiumN,N-di(carboxymethyl)-N-carbodithioate), MGDTC (sodiumN-methyl-D-glucamine-N-carbodithioate), MeOBGDTC (sodiumN-(4-methoxybenzyl)-D-glucamine-N-carbodithioate), DEDTC (sodiumN,N-diethyl-N-carbodithioate), Di-PDTC (sodiumN,N-diisopropyl-N-carbodithioate), and NAC is (N-acetyl cysteine). Theleast effective compounds were MeOBGDTC and NAC.

EXAMPLE 6

PDTC Inhibits TNF Induction of VCAM-1/VLA-4 Mediated Adhesion

In order to define whether PDTC inhibition of VCAM-1 regulation isassociated with functional consequences, the binding of Molt-4 cells toHUVEC cells either unstimulated or stimulated with TNFa (100 U/ml) forsix hours in the presence or absence of PDTC was examined. Molt-4 cellshave been previously shown to bind to activated HUVEC via a VCAM-1dependent mechanism. As shown in FIG. 6, the percentage of Molt-4binding to HUVEC cells decreased when PDTC was present in the media.

EXAMPLE 7

PDTC Inhibits Monocyte Binding to the Thoracic Aorta of Cholesterol FedRabbits

An experiment was performed to determine whether the thiol antioxidantPDTC would be efficacious in blocking the first monocyte bindingcomponent of atherosclerosis in an experimental animal model. One matureNew Zealand white rabbit (3.5 Kg) received an intravenous injection ofPDTC (20 mg/Kg, as a concentration of 20 mg/ml in PBS) once daily for 5days. Injections were given via an indwelling cannula in the marginalear vein, which was kept patent by flushing with heparinized salinesolution. The PDTC solution was mixed fresh daily or on alternate days(stored light-protected at 4° C.), and filtered (0.45 mm pore filter)just prior to use. After the first injection, when the cannula wasplaced, the drug was administered with the rabbit in the conscious statewithout apparent discomfort or other ill effect. On the second day ofinjections, the rabbit was given chow containing 1% cholesterol byweight, which was continued throughout the remainder of the experiment.On the fifth day, the animal was euthanized and the thoracic aorta wasexcised and fixed. After appropriate preparation, the sample was imagedon the lower stage of an ISI DS-130 scanning electron microscopeequipped with a LaB emitter. Using dual-screen imaging and a transparentgrid on the CRT screen, 64 adjacent fields at a 620× magnification wereassessed, to cover an area of approximately 1.3 mm². Within each field,the number of adherent leukocytes (WBC) and erythrocytes (RBC) werecounted and recorded.

The data from the arch sample are as follows: 5 WBC and -25 RBC per 1.3mm² field. This level of WBC adhesion is similar to control animals fedregular chow (about 7 per field have been seen in arch and thoracicsamples from 2 `negative control` experiments). `Positive control`rabbits fed 1% cholesterol for 4 days but not given antioxidant showabout a 5-fold increase in adhesion, to 38 WBC/1.3 mm². A considerableamount of mostly cell-sized debris was observed adherent to each archsample. It is unclear whether this material is an artifact ofpreparation, or was present in vivo, and if so, whether it is related toPDTC administration. These studies suggest that PDTC infusions caneffectively block initial monocyte adhesion to the aortic endothelium.

III. Pharmaceutical Compositions

Humans, and other animals, in particular, mammals, suffering fromcardiovascular disorders, and other inflammatory conditions mediated byVCAM-I can be treated by administering to the patient an effectiveamount of one or more of the above-identified compounds or apharmaceutically acceptable derivative or salt thereof in apharmaceutically acceptable carrier or diluent. The active materials canbe administered by any appropriate route, for example, orally,parenterally, intravenously, intradermally, or subcutaneously.

As used herein, the term pharmaceutically acceptable salts or complexesrefers to salts or complexes that retain the desired biological activityof the above-identified compounds and exhibit minimal undesiredtoxicological effects. Nonlimiting examples of such salts are (a) acidaddition salts formed with inorganic acids (for example, hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, andthe like), and salts formed with organic acids such as acetic acid,oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid,benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, naphthalenedisulfonic acid, andpolygalacturonic acid; (b) base addition salts formed with polyvalentmetal cations such as zinc, calcium, bismuth, barium, magnesium,aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and thelike, or with an organic cation formed fromN,N-dibenzylethylene-diamine, D-glucosamine, ammonium,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount without causing serious toxic effectsin the patient treated. A preferred dose of the active compound for allof the above-mentioned conditions is in the range from about 0.5 to 500mg/kg, preferably 1 to 100 mg/kg per day. The effective dosage range ofthe pharmaceutically acceptable derivatives can be calculated based onthe weight of the parent compound to be delivered. If the derivativeexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the derivative, or by other means known tothose skilled in the art.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing 1 to 3000 mg,preferably 5 to 500 mg of active ingredient per unit dosage form. A oraldosage of 25-250 mg is usually convenient.

The active ingredient should be administered to achieve peak plasmaconcentrations of the active compound of about 0.1 to 100 μM, preferablyabout 1-10 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The active compound or pharmaceutically acceptable salt or derivativethereof can be administered as a component of an elixir, suspension,syrup, wafer, chewing gum or the like. A syrup may contain, in additionto the active compounds, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable derivatives or saltsthereof can also be administered with other active materials that do notimpair the desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, orantiviral compounds. The active compounds can be administered with lipidlowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as varapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalopril, and β-blockers such as propanalol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteroidal antiinflammatories such as ibuprofen, indomethacin,aspirin, fenoprofen, mefenamic acid, flufenamic acid, sulindac. Thecompound can also be administered with corticosteriods.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediarninetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

The active compound can also be administered through a transdermalpatch. Methods for preparing transdermal patches are known to thoseskilled in the art. For example, see Brown, L., and Langer, R.,Transdermal Delivery of Drugs, Annual Review of Medicine, 39:221-229(1988), incorporated herein by reference.

In another embodiment, the active compounds are prepared with carriersthat will protect the compound against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound or its monophosphate, diphosphate, and/or triphosphatederivatives are then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description of theinvention. Such modifications and variations are intended to come withinthe scope of the appended claims.

We claim.
 1. A method for the treatment of cardiovascular disease inhumans comprising administering to the human an effective amount of adithiocarbamate of the formula:

    R.sup.1 SC(S)NR.sup.2 R.sup.3

wherein R¹ is H, sodium, potassium, or NR⁴ R⁵ R⁶ R⁷, wherein R⁴, R⁵, R⁶,and R⁷ are independently hydrogen, C₁₋₆ linear, branched, or cyclicalkyl, hydroxy(c₁₋₆) alkyl, or aryl, and R² and R³ are independentlyC₁₋₁₀ linear, branched or cyclic alkyl; --(CHOH)_(n) (CH₂)_(n) OH,wherein n is 0-6; --(CH₂)_(n) CO₂ R¹, --(CH₂)_(n) CO₂ R⁴ ;hydroxy(C₁₋₆)alkyl--, or R² or R³ together constitute a bridge of theformula --(CH₂)_(m) --, wherein m is 3-6, and wherein R⁴ is alkyl, aryl,alkylaryl, or aralkyl.
 2. The method of claim 1, wherein thedithiocarbamate is selected from the group consisting of sodiumpyrrolidine-N-carbodithioate, sodium N-methyl -N- carboxymethyl-N-carbodithioate, trisodium N,N-di (carboxymethyl) -N-carbodithioate,sodium N-methyl-D-glucamine-N-carbodithioate, sodiumN,N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate) , andsodium N,N-diisopropyl-N-carbodithioate.
 3. The method of claim 1,wherein the dithiocarbamate is sodium pyrrolidine-N-carbodithioate. 4.The method of claim 1, wherein the cardiovascular disease isatherosclerosis.
 5. The method of claim 1, wherein the cardiovasculardisease is post-angioplasty restenosis.
 6. The method of claim 1,wherein the cardiovascular disease is coronary artery disease.
 7. Themethod of claim 1, wherein the cardiovascular disease is angina.
 8. Themethod of claim 1, wherein the cardiovascular disease is a small vesseldisease.
 9. The method of claim 1, wherein the dithiocarbamate isadministered in a dosage of between 0.5 and 500 mg/kg body weight. 10.The method of claim 1, wherein the dithiocarbamate is administered byperfusion balloon catheter.
 11. The method of claim 1, wherein thedithiocarbamate is administered in combination with a pharmaceuticalagent selected from the group consisting of a lipid lowering agent, aplatelet aggregation inhibitor, an antithrombotic agent, a calciumchannel blocker, an angiotensin converting enzyme (ACE) inhibitor, aS-blocker, a nonsteroidal antiinflammatory, and a corticosteroid. 12.The method of claim 1, wherein the ester moiety of --(CH₂)_(n) CO₂ R⁴ isselected from the group consisting of acetyl, propionyl, and butyryl.